HIV-1 has been identified as the etiologic agent of the acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., Science 220, 868-871, 1983; Popovic et al, Science 224, 497-500, 1984; Gallo et al., Science 224, 500-503, 1984). Infected individuals generally develop antibodies to the virus within several months of exposure (Sarngadharan et al., Science 224, 506-508, 1984), which has made possible the development of immunologically based tests which can identify most of blood samples from infected individuals. This is a great advantage in diagnosis, and is vital to maintaining the maximum possible safety of samples from blood banks.
An important aspect of HIV-1 is its genetic variability (Hahn et al., Proc. Natl. Acad. Sci. U.S.A. 82, 4813-4817, 1985). This is particularly evident in the gene for the outer envelope glycoprotein (Starcich et al., Cell 45, 637-648, 1986; Alizon et al., Cell 46, 63-74, 1986; Gurgo et al., Virology 164, 531-536, 1988). Since the outer envelope glycoprotein is on the surface of the virus particle and the infected cell, it is potentially one of the primary targets of the immune system, including the target of neutralizing antibodies and cytotoxic T cells. This variability may also lead to differences in the ability of antigens from different strains of HIV-1 to be recognized by antibodies from a given individual, as well as to differences in the ability of proteins from different strains of virus to elicit an immune response which would be protective against the mixture of virus strains that exists in the at risk populations.
Several biologically active complete molecular clones of various strains of HIV-1 have been obtained and sequenced. These clones, however, seem to represent viral genotypes which are relatively atypical of United States HIV-1 isolates. In addition, several of the translational reading frames for non-structural viral proteins are not complete. Further, viruses derived from these clones do not grow in macrophages, in contrast to many HIV-1 field isolates and, perhaps, because of this lack of ability to infect macrophage efficiently, these clones do not replicate well in chimpanzees. This latter ability is important for testing candidate vaccines in animal systems. In addition, the ability to infect macrophages is critical in evaluating the possible protective efficacy of an elicited immune response since neutralization of infectivity on macrophages may differ from the better studied neutralization on T cells.
Neutralizing antibodies (Robert-Guroff et al., Nature 316, 72-74, 1985; Weiss et al., Nature 316, 69-72, 1985) have been demonstrated in infected individuals, as have cytotoxic T cell responses (Walker et al, Nature 328, 345-348, 1988). Although these do not appear to be protective, it is likely that if they were present prior to infection, they would prevent infection, especially by related strains of virus. This is supported by the finding that macaques can be protected by immunization with inactivated simian immunodeficiency virus (SIV) from infection with the homologous live virus (Murphy-Corb et al., Science 246, 1293-1297, 1989). Chimps also have been passively protected against challenge by live virus by prior administration of neutralizing antibodies to the same virus (Emini et al., J. Virol. 64, 3674-3678, 1989). One problem, however, is that at least some of the neutralizing antibodies studied depend on recognition of a variable region on the envelope (Matsushita et al., J. Virol. 62, 2107-2114, 1988; Rusche et al., Proc. Natl. Acad. Sci. U.S.A. 85, 3198-3202, 1988; Skinner et al., AIDS Res. Hum. Retroviruses 4, 187-197, 1988) called the V3 region (Starcich et al., Cell 45, 637-648, 1986).
An at least partial solution to the problem of viral heterogeneity is to identify prototypical HIV-1 strains, that is, those that are most similar by DNA sequence data or serologic reactivity to strains present in the population at risk. The inclusion of a limited number of such prototype strains in a polyvalent vaccine cocktail might then result in elicitation of an immune response protective against most naturally occurring viruses within a given population. Such a mixture should also provide the maximum possible sensitivity in diagnostic tests for antibodies in infected individuals.
Components of highly representative isolates of a geographical area provide the maximum possible sensitivity in diagnostic tests and vaccines. Production of viral proteins from molecular clones by recombinant DNA techniques is the preferred and safest means to provide such proteins. Molecular clones of prototype HIV-1 strains can serve as the material from which such recombinant proteins can be made. The use of recombinant DNA avoids any possibility of the presence of live virus and affords the opportunity of genetically modifying viral gene products. The use of biological active clones ensures that the gene products are functional and hence, maximizes their potential relevance.
Infectious clones, that is, those which after transfection into recipient cells produce complete virus, are desirable for several reasons. One reason is that the gene products are by definition functional; this maximizes their potential relevance to what is occurring in vivo. A second reason is that genetically altered complete virus is easy to obtain. Consequently, the biological consequences of variability can be easily assessed. For example, the effect of changes in the m envelope gene on the ability of the virus to be neutralized by antibody can be easily addressed. Using this technique, a single point mutation in the envelope gene has been shown to confer resistance to neutralizing antibody (Reitz et al., Cell 54, 57-63, 1988). A third reason is that a clonal virus population provides the greatest possible definition for challenge virus in animals receiving candidate vaccines, especially those including components of the same molecularly cloned virus.